Recently, an ultra-high throughput sequencing system based on pyrophosphate sequencing was disclosed which allows for the sequencing of a bacterial genome in essentially not more than one week (WO 04/70007, WO 05/03375, Margulies, M., et al., Nature 437 (2005) 376-80). Starting from sheared genomic DNA, single molecule fragments are bound to beads which are captured in a PCR-reaction-mixture-in-oil emulsion. Amplification then results in a library of clonally amplified DNA with each bead carrying multiple copies of the same fragment.
After breakage of the emulsion and denaturation of the PCR products into single strands, beads are deposited into the multiple wells of a fiber-optic picotiter plate such that one well carries not more than a single bead. Subsequently, in a sequencing by synthesis reaction, a primer extension reaction is performed, where the 4 different A, G, C, and T nucleoside triphosphates or their respective analogs are supplied in a repetitive series of events and the sequence of the nascent strand is inferred from chemical products derived from the extension reaction catalyzed by the DNA polymerase. In particular, the sequencing by synthesis reaction is a pyrophosphate sequencing reaction, characterized in that generation of pyrophosphate is detected as follows:PPi+adenosine 5′ phosphosulfate (APS)→ATP, catalyzed in the presence of ApyraseATP+luciferin→light+oxy luciferin, catalyzed in the presence of LuciferaseDetection of luminescence of oxyluciferin
With the ultra high throughput sequencing system as disclosed in WO 04/70007 and WO 05/03375, more than 1,000,000 pyrophosphate sequencing reactions can be carried out simultaneously. The generation of Pyrophosphate is triggering a luminescent reaction cascade and light is finally with a CCD camera.
With respect to this technology, WO 04/69849 discloses a method of amplifying a plurality of nucleic acids (e.g., each sequence of a DNA library, transcriptome, or genome) in a rapid and economical manner in a single reaction tube. More particularly, WO 04/69849 discloses a simultaneous clonal amplification (e.g., by PCR) of a plurality of samples (as many as several hundred thousand) in one reaction vessel. In this contest, WO 04/69849 provides a means for encapsulating a plurality of DNA samples individually in a microcapsule of an emulsion (i.e., a microreactor), performing amplification of the plurality of encapsulated nucleic acid samples simultaneously, and releasing said amplified plurality of DNA from the microcapsules for subsequent reactions. For example, single copies of the nucleic acid template species are hybridized to capture beads comprising, e.g., capture oligonucleotides or chemical groups that bind to the nucleic acid template. The beads are suspended in complete amplification solution and emulsified to produce microreactors (typically 100 to 200 microns in diameter). After this, amplification (e.g., PCR) is used to clonally increase copy number of the initial template species in the microreactors, and these copies bind to the capture beads in the microreactors. Alternatively, capture beads are added to an amplification reaction mixture comprising nucleic acid template and this mixture is emulsified to produce microreactors. Amplification (e.g., PCR) is used to clonally increase copy number of the initial template species in the microreactors, and these copies bind to the capture beads in the microreactors. Thus, the microreactors according to WO 05/03375 allow the simultaneous clonal and discrete amplification of many different templates without cross contamination of the amplified products or reagents, or domination of one particular template or set of templates (e.g., PCR bias).
However, according to WO 05/03375 it is necessary to perform an adaptor ligation step in which a plurality of different nucleic acid molecules is tagged with an adaptor sequence which may subsequently become bound to a bead with a covalently attached complementary adaptor sequence.
Another important DNA analysis technique is analytical polymerase chain reaction (PCR). Quantification of PCR, however, to date is based on analogous measurement modes. In some cases, however, a digital counting principle would be highly desirable for example in areas such as cancer detection: quantification of mutant alleles in an excess wild type background, detection of allelic imbalance, gene expression of rare transcripts and/or mutant alleles in transcripts, and viral detection and quantification.
Thus, the availability of a digital counting of DNA/cDNA/mRNA addresses unserved needs in molecular medicine, e.g., highly relevant for cancer diagnostics, circulating tumor cells, pre-natal embryonic cells where a specific event has to be detected within a high background.
It was therefore an object of the present invention to provide an improved method and improved reagents for the simultaneous analysis of multiple nucleic acid molecules.
In a particular aspect it was an object of the present invention to provide a solid support or a plurality of solid supports which may be used to improve the sequencing workflow described above or enable digital PCR counting.
Various solid supports comprising immobilized nucleic acids such as beads comprising immobilized oligonuicleotides are well known in the art. The exact design and configuration of these beads depends on the application for which they are used: Steinberg-Tatman, G., et al. (Bioconjugate Chemistry 17 (2006) 841-848) describe a method for synthesizing beads with two different oligonucleotides attached to the surface via a non cleavable linker One oligonucleotide is used as a sequence specific capture probe the other as decoding sequence.
U.S. Pat. No. 5,639,603 describes beads with on ore more immobilized oligonucleotide decoding tags.
Xu, Xiaoyang; Rosi, Nathaniel, L.; Wang, Yuhuang; Huo, Fengwei; Mirkin, Chad A. Journal of the American Chemical Society 128(29) (2006) 9286-9287 describe gold particles with two different oligonucleotides attached to the surface for building up ordered mulitparticle nanostructures.
WO 2001/062982 and U.S. Pat. No. 5,641,658 describe PCR on bead surfaces with two immobilized primers. The PCR method is named bridge amplification.
WO 2001/012862 describes a method for generating a pool of oligonucleotides by cleaving different oligonucleotides which are attached to a substrate via different cleavable linkers.
WO 2007/111937 describes an array for primer pairs where at least one primer is attached via a cleavable linkage for enrichment of genomic DNA used in sequencing.
KR 2007044677 describes beads with a first and second-immobilized PCR primers primer for use in emulsion PCR. The first primer is non cleavable. The release of the second primer is achieved by changing the pH value. Yet, changing the pH value has the disadvantage of potentially undesired side reactions and furthermore makes further processing of the sample more difficult.